The lack of sensitive, specific, multiplex-able assays for most human proteins is a major technical barrier that impedes progress in the biomedical sciences by prohibiting hypothesis testing in quantitative proteomics, where relationships between protein abundance and biology are sought. If a robust, economical, and widely diffused capability to measure all human proteins existed, the research community would have the collective means to assess the utility of all human proteins as biomarkers in hundreds of diseases and biological processes in the most efficient way. This would likely have a profound impact on healthcare costs and outcomes. Multiple reaction monitoring mass spectrometry (MRM-MS) has been used for decades in clinical reference laboratories for measurement of small molecules in plasma, such as drug metabolites or metabolites that accumulate as a result of inborn errors of metabolism. More recently, MRM-MS (coupled to stable isotope dilution) has been adapted to measure the concentration of candidate protein biomarkers in plasma and cultured cell lysates, using proteotypic peptides as specific stoichiometric surrogates. This proposal is a pilot study to assess feasibility and scalability of a human proteome detection and quantitation project (hPDQ), the goal of which is to enable individual researchers to measure large collections of human proteins in biological samples with absolute specificity, at throughput and cost levels that permit the study of meaningfully large biological populations (500-5,000 samples). To establish feasibility and scalability, we will generate a dataset of unprecedented scale, configuring 800 novel assays to measure 400 human proteins (2% of the basic, unmodified human proteome), and demonstrating the performance of these assays in a panel of ca. 50 genomically well characterized breast cancer cell lines. Aim 1. Select 400 cancer-related proteins. Aim 2. Configure 800 multiplexed LC-MRM-MS assays targeting the 400 cancer-relevant proteins (2 peptides per protein). Aim 3. Demonstrate the ability of the multiplexed assays to measure 400 cancer-relevant proteins in a panel of 51 genomically well characterized breast cancer cell lines. PUBLIC HEALTH RELEVANCE: The human body makes many thousands of proteins. Abnormal changes in the abundances or activities of these proteins are responsible for virtually all human disease. Due to longstanding technological limitations, we only have the ability to measure a very small number of human proteins, and this has limited our ability to find causes of diseases such as cancer, to develop better diagnostic tests, and to develop better therapies. Our research aims to use a new technology to generate assays to measure large numbers of human proteins in cancer cells. Success of this project will greatly facilitate translation of basic research into tangible medical benefit for patients.